Project Summary Despite early detection and adjuvant therapy, breast cancer remains the leading cause of cancer mortality in women, largely due to distant, incurable recurrences arising years, or even decades, after treatment of the primary tumor. These recurrent, metastatic tumors arise from the pool of residual local and disseminated tumor cells (DTCs) that survive primary treatment and remain in the host in a presumed dormant state. Indeed, the presence of bone marrow DTCs following treatment is independently associated with a substantially increased risk of recurrence. At present, however, the mechanisms enabling residual tumor cells to maintain dormancy and ultimately recur are poorly understood, and DTC-directed surveillance and treatment approaches are non-existent. Consequently, the ability to biologically characterize, accurately measure and therapeutically target dormant DTCs would be a transformational new approach to preventing recurrence. We hypothesize that disabling the survival mechanisms employed by dormant DTCs will reduce tumor recurrence and thereby improve survival. Using genetically engineered mouse models that faithfully recapitulate tumor dormancy and recurrence, we have discovered that autophagy and mTOR signaling are each critical to the survival of DTCs, and that agents inhibiting these pathways deplete the reservoir of dormant residual tumor cells, thereby preventing tumor recurrence. The objective of this proposal is to translate these biological insights and preclinical therapeutic data to generate the interventional approach, requisite laboratory assays, and demonstration of feasibility, safety and clinical efficacy of targeting DTCs that will be required for large-scale, definitive clinical trials and surveillance studies. The specific aims of this application are to: 1) Perform a proof-of-concept clinical trial of everolimus (EVE, targeting mTOR), and hydroxychloroquine (HCQ, targeting autophagy) in women with detectable DTCs after primary treatment; and 2) Employ preclinical mouse models to concurrently optimize therapeutic approaches and advance discoveries for the eradication of DTCs. The randomized, open-label pilot trial in Aim 1 will investigate the feasibility and safety of HCQ, EVE or the combination, and their effects on DTC burden. We will also refine and validate a novel flow cytometric assay to improve the sensitivity of detection, enumeration and molecular characterization of the DTC biomarker. In Aim 2, a co-clinical trial in mice will optimize the effects of HCQ, EVE, and their combination, investigate critical parameters of these effects necessary to inform clinical trials, and extend these models to more closely reflect clinical treatment. In addition, molecular phenotyping of residual tumor cells will uncover additional targets for future trials. Ultimately, the ability to identify, enumerate and therapeutically target DTCs has the potential to transform surveillance and treatment options for breast cancer survivors and prevent women from succumbing to this deadly disease.